This invention relates to methods for assaying for the presence of the hexose sugar, inositol, and to methods for the production of antibodies to inositol derivatives. This invention provides quantitative assays for the various isomers of this compound.
Inositol is of interest in a number of biological systems. The D-chiro isomer of this compound has been shown to be useful both as an indicator of insulin resistant diabetes and as a therapeutic agent for this disorder. See, U.S. Pat. No. 5,124,360.
Myoinositol phosphates are known to be involved in the modulation of Ca.sup.2+ homeostasis (Hughes, A. R. et al. Am. Rev. Resp. Dis. 141 (3 Pt 2) S115-8 (March, 1990)), have been implicated in the mechanism of malignant hypothermia (Scholz, J. et al. Br. J. Anesth. Vol. 66, pp. 692-6 (1991)) and have a significant role in many other physiological systems. Myoinositol phosphates can be quantified following their enzymatic conversion to myoinositol e.g. with alkaline phosphatase.
Direct chemical assays for inositol phosphates are known. However, these cannot distinguish between inositol isomers (optical techniques), lack sensitivity (gas chromatography and nuclear magnetic resonance), are very expensive (fast atom bombardment), or are specific for inositol-1,4,5-triphosphate (Palmer S. and Wakelam M. J. Biochimica Et Biophysica Acta. 1014(3):239-46, 1989).
Immunoassays per se have long been known. Unfortunately, such methods are not without their limitations. The success of an immunoassay rests upon the ability of an animal immune system to recognize a substance as foreign to the normal and to generate antibodies which are specific to the analyte of interest. Often, a compound of interest is simply too small to permit the animal immune system to recognize it as a foreign substance. Sometimes, the analyte is, in fact, not foreign at all. In still other cases, antibodies can be produced, but they are not very specific and cross react with compounds other than the analyte.
An analyte can sometimes be linked to a larger molecule so that the resulting conjugated compound is large enough to be recognized by the animal immune system as foreign. In this circumstance, when the analyte is linked to a large, relatively inert substance (such as bovine serum albumin), it is referred to as a hapten. The conjugated hapten is injected into an animal (such as a rabbit) with the hope that antibodies to the hapten will be produced and that they will be specific to the hapten. Unfortunately, attempts to use this technique to create an immunoassay for inositols have not been successful.